Droplet Actuator Devices and Methods Employing Magnetic Beads

ABSTRACT

A method comprising effecting a change in a shape of a droplet, wherein the droplet is disposed over a substrate in sensing proximity to a sensor and the droplet has a starting surface area exposed to the sensor; and producing an expanded surface area of the droplet in the sensing proximity exposed to the sensor, wherein the expanded surface area exposed to the sensor is greater than the starting surface area exposed to the sensor.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/719,731, filed on May 22, 2015, entitled “Droplet Actuator Devicesand Methods Employing Magnetic Beads,” which is a continuation of U.S.application Ser. No. 12/524,495 (now U.S. Pat. No. 9,046,514), filed onJul. 24, 2009, entitled “Droplet Actuator Devices and Methods EmployingMagnetic Beads,” which is a U.S. National Stage entry of PCT ApplicationNo. PCT/US2008/053545, filed on Feb. 11, 2008, entitled “DropletActuator Devices and Methods Employing Magnetic Beads,” which claimspriority to provisional U.S. Patent Application No. 60/900,653, filed onFeb. 9, 2007, entitled “Immobilization of magnetically-responsive beadsduring droplet operations”; 60/980,772, filed on Oct. 17, 2007, entitled“Immobilization of magnetically-responsive beads in droplets”;60/969,736, filed on Sep. 4, 2007, entitled “Droplet actuator assayimprovements”; and 60/980,762, filed Oct. 17, 2007, entitled “Dropletactuator assay improvements;” each of the aforementioned disclosures isincorporated herein by reference in its entirety.

GRANT INFORMATION

This invention was made with government support under CA114993-01A2 andDK066956-02 awarded by the National Institutes of Health of the UnitedStates. The United States Government has certain rights in theinvention.

FIELD OF THE INVENTION

The invention relates generally to the field of droplet actuators, andin particular, to droplet actuators configured for conducting dropletbased protocols requiring droplet operations to be conducted usingdroplets comprising beads, especially magnetically responsive beads. Theinvention also relates to methods of making and using such dropletactuators.

BACKGROUND OF THE INVENTION

Droplet actuators are used to conduct a wide variety of dropletoperations. A droplet actuator typically includes a substrate associatedwith electrodes for conducting droplet operations on a dropletoperations surface thereof and may also include a second substratearranged in a generally parallel fashion in relation to the dropletoperations surface to form a gap in which droplet operations areeffected. The gap is typically filled with a filler fluid that isimmiscible with the fluid that is to be subjected to droplet operationson the droplet actuator.

In some applications of a droplet actuator there is a need for using“beads” for various assays. For protocols that make use of beads, thebeads are typically used to bind to one or more target substances in amixture of substances. The target substances may, for example, beanalytes or contaminants. There is a need for an efficient approach tobead washing on a droplet actuator in order to reduce the amount of oneor more substances in a bead-containing droplet that may be in contactwith or exposed to the surface of the bead or beads.

BRIEF DESCRIPTION OF THE INVENTION

One aspect of the invention provides efficient immobilization ofmagnetically responsive beads for droplet operations that make use ofmagnetically responsive beads in droplet-based applications. Examplesinclude assays that require execution of bead washing protocols, such aspyrosequencing and immunoassay applications. In one example, theinvention provides techniques employing magnetic forces for immobilizingmagnetically responsive beads during droplet splitting operations. Thetechniques of the invention are particularly useful in protocols forwashing magnetically responsive beads within a droplet actuator. Amongother advantages, the techniques of the invention avoid undue clumpingor aggregation of the magnetically responsive beads. During dropletsplitting operations, the techniques can usefully immobilizesubstantially all of the magnetically responsive beads within a droplet.Techniques of the invention may ensure immobilization and retention ofsubstantially all magnetically responsive beads during a droplet washingoperation. Upon completion of a washing process, the techniques of theinvention ensure resuspension of substantially all of the magneticallyresponsive beads within the liquid and with substantially no clumping oraggregation thereof.

Another aspect of the invention provides improved droplet actuators andrelated methods for effecting improved droplet-based assay operations.Embodiments of the invention provide mechanisms for reducing thecrossover of magnetic fields within a droplet actuator. Otherembodiments of the invention provide mechanisms for reducing thecarryover of beads and other substances within a droplet actuator. Yetother embodiments of the invention provide mechanisms for improvingdroplet detection operations within a droplet actuator.

Definitions

As used herein, the following terms have the meanings indicated.

“Activate” with reference to one or more electrodes means effecting achange in the electrical state of the one or more electrodes whichresults in a droplet operation.

“Bead,” with respect to beads on a droplet actuator, means any bead orparticle that is capable of interacting with a droplet on or inproximity with a droplet actuator. Beads may be any of a wide variety ofshapes, such as spherical, generally spherical, egg shaped, disc shaped,cubical and other three dimensional shapes. The bead may, for example,be capable of being transported in a droplet on a droplet actuator orotherwise configured with respect to a droplet actuator in a mannerwhich permits a droplet on the droplet actuator to be brought intocontact with the bead, on the droplet actuator and/or off the dropletactuator. Beads may be manufactured using a wide variety of materials,including for example, resins, and polymers. The beads may be anysuitable size, including for example, microbeads, microparticles,nanobeads and nanoparticles. In some cases, beads are magneticallyresponsive; in other cases beads are not significantly magneticallyresponsive. For magnetically responsive beads, the magneticallyresponsive material may constitute substantially all of a bead or onecomponent only of a bead. The remainder of the bead may include, amongother things, polymeric material, coatings, and moieties which permitattachment of an assay regent. Examples of suitable magneticallyresponsive beads are described in U.S. Patent Publication No.2005-0260686, entitled, “Multiplex flow assays preferably with magneticparticles as solid phase,” published on Nov. 24, 2005, the entiredisclosure of which is incorporated herein by reference for its teachingconcerning magnetically responsive materials and beads. The beads mayinclude one or more populations of biological cells adhered thereto. Insome cases, the biological cells are a substantially pure population. Inother cases, the biological cells include different cell populations,e.g., cell populations which interact with one another.

“Droplet” means a volume of liquid on a droplet actuator that is atleast partially bounded by filler fluid. For example, a droplet may becompletely surrounded by filler fluid or may be bounded by filler fluidand one or more surfaces of the droplet actuator. Droplets may take awide variety of shapes; nonlimiting examples include generally discshaped, slug shaped, truncated sphere, ellipsoid, spherical, partiallycompressed sphere, hemispherical, avoid, cylindrical, and various shapesformed during droplet operations, such as merging or splitting or formedas a result of contact of such shapes with one or more surfaces of adroplet actuator.

“Droplet operation” means any manipulation of a droplet on a dropletactuator. A droplet operation may, for example, include: loading adroplet into the droplet actuator; dispensing one or more droplets froma source droplet; splitting, separating or dividing a droplet into towor more droplets; transporting a droplet from one location to another inany direction; merging or combining two or more droplets into a singledroplet; diluting a droplet; mixing a droplet; agitating a droplet;deforming a droplet; retaining a droplet in position; incubating adroplet; heating a droplet; vaporizing a droplet; cooling a droplet;disposing of a droplet; transporting a droplet out of a dropletactuator; other droplet operations described herein; and/or anycombination of the foregoing. The terms “merge,” “merging,” “combine,”“combining” and the like are used to describe the creation of onedroplet from two or more droplets. It should be understood that whensuch a term is used in reference to two or more droplets, anycombination of droplet operations sufficient to result in thecombination of the two or more droplets into one droplet may be used.For example, “merging droplet A with droplet B,” can be achieved bytransporting droplet A into contact with a stationary droplet B,transporting droplet B into contact with a stationary droplet A, ortransporting droplets A and B into contact with each other. The terms“splitting,” “separating” and “dividing” are not intended to imply anyparticular outcome with respect to size of the resulting droplets (i.e.,the size of the resulting droplets can be the same or different) ornumber of resulting droplets (the number of resulting droplets may be 2,3, 4, 5 or more). The term “mixing” refers to droplet operations whichresult in more homogenous distribution of one or more components withina droplet. Examples of “loading” droplet operations includemicrodialysis loading, pressure assisted loading, robotic loading,passive loading, and pipette loading.

“Immobilize” with respect to magnetically responsive beads, means thatthe beads are substantially restrained in position in a droplet or infiller fluid on a droplet actuator. For example, in one embodiment,immobilized beads are sufficiently restrained in position to permitexecution of a splitting operation on a droplet, yielding one dropletwith substantially all of the beads and one droplet substantiallylacking in the beads.

“Magnetically responsive” means responsive to a magnetic field.“Magnetically responsive beads” include or are composed of magneticallyresponsive materials. Examples of magnetically responsive materialsinclude paramagnetic materials, ferromagnetic materials, ferromagneticmaterials, and metamagnetic materials. Examples of suitable paramagneticmaterials include iron, nickel, and cobalt, as well as metal oxides,such as Fe.sub.3O.sub.4, BaFe.sub.12O.sub.19, CoO, NiO, Mn.sub.2O.sub.3,Cr.sub.2O.sub.3, and CoMnP.

“Washing” with respect to washing a magnetically responsive bead meansreducing the amount and/or concentration of one or more substances incontact with the magnetically responsive bead or exposed to themagnetically responsive bead from a droplet in contact with themagnetically responsive bead. The reduction in the amount and/orconcentration of the substance may be partial, substantially complete,or even complete. The substance may be any of a wide variety ofsubstances; examples include target substances for further analysis, andunwanted substances, such as components of a sample, contaminants,and/or excess reagent. In some embodiments, a washing operation beginswith a starting droplet in contact with a magnetically responsive bead,where the droplet includes an initial amount and initial concentrationof a substance. The washing operation may proceed using a variety ofdroplet operations. The washing operation may yield a droplet includingthe magnetically responsive bead, where the droplet has a total amountand/or concentration of the substance which is less than the initialamount and/or concentration of the substance. Other embodiments aredescribed elsewhere herein, and still others will be immediatelyapparent in view of the present disclosure.

The terms “top” and “bottom” are used throughout the description withreference to the top and bottom substrates of the droplet actuator forconvenience only, since the droplet actuator is functional regardless ofits position in space.

When a given component, such as a layer, region or substrate, isreferred to herein as being disposed or formed “on” another component,that given component can be directly on the other component or,alternatively, intervening components (for example, one or more coating,layers, interlayers, electrodes or contacts) can also be present. Itwill be further understood that the terms “disposed on” and “formed on”are used interchangeably to describe how a given component is positionedor situated in relation to another component. Hence, the terms “disposedon” and “formed on” are not intended to introduce any limitationsrelating to particular methods of material transport, deposition, orfabrication.

When a liquid in any form (e.g., a droplet or a continuous body, whethermoving or stationary) is described as being “on”, “at”, or “over” anelectrode, array, matrix or surface, such liquid could be either indirect contact with the electrode/array/matrix/surface, or could be incontact with one or more layers or films that are interposed between theliquid and the electrode/array/matrix/surface.

When a droplet is described as being “on” or “loaded on” a dropletactuator, it should be understood that the droplet is arranged on thedroplet actuator in a manner which facilitates, using the dropletactuator to conduct one or more droplet operations on the droplet, thedroplet is arranged on the droplet actuator in a manner whichfacilitates sensing of a property of or a signal from the droplet,and/or the droplet has been subjected to a droplet operation on thedroplet actuator.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate first and second top views, respectively, ofa portion of a droplet actuator in use during a first and second phase,respectively, of a droplet splitting operation;

FIGS. 2A and 2B illustrate a side and top views of a section of adroplet actuator that includes a magnet that is place at a positionwhich is array from the splitting zone;

FIG. 3 illustrates a side view of a section of a droplet actuator thatincludes two magnets that are arranged above and below droplet;

FIGS. 4A and 4B illustrate side views of a section of a droplet actuatorthat includes four magnets that arranged at positions surrounding thedroplet;

FIGS. 5A and 5B illustrate a first and second top view, respectively, ofa section of a droplet actuator during a first and second phase,respectively, of a droplet splitting operation;

FIG. 6 illustrates a side view of a section of a droplet actuator thatincludes a magnetic shield for reducing the crossover of magneticfields;

FIG. 7 illustrates a top view of a section of a droplet actuator thatincludes a magnet having poles that are oppositely arranged for reducingthe crossover of magnetic fields;

FIG. 8 illustrates a map of magnetic fields that are arranged in arelationship such as the one shown in FIG. 7;

FIG. 9 illustrates a top view of a section of a droplet actuatorarranged to reduce carryover at a droplet detection region;

FIGS. 10A and 10B illustrate a top view of a section of a dropletactuator configured to reduce carryover;

FIGS. 11A, 11B, and 11C illustrate side views of section of a dropletactuator configured for improving the sensitivity of droplet detection;

FIG. 12 illustrates a top view of a section of a droplet actuatorconfigured for improving the sensitivity of droplet detection;

FIG. 13 illustrates a side view of a section of a droplet actuatorconfigured for improving the sensitivity of droplet detection;

FIGS. 14A and 14B illustrate top views of a modular droplet actuatorassembly, which provides a universal assembly for orienting a magnetassembly to a droplet actuator; and

FIG. 15 illustrates a side view of a modular droplet actuator assembly,which is another nonlimiting example of a universal assembly fororienting a magnet assembly to a droplet actuator.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates, among other things, to droplet actuatorsconfigured for immobilizing magnetically-responsive beads and to methodsof making and using such droplet actuators. As an example, the dropletactuators are useful for immobilizing beads in droplets on the dropletactuators, thereby facilitating the execution of protocols which requireimmobilization of such beads, sued as bead washing protocols. Theinvention also provides techniques for reducing or eliminating carryoverof substances from droplet to droplet in a droplet actuator andtechniques for maximizing signal detection on a droplet actuator.

8.1 Bead Loss During Droplet Splitting

FIGS. 1A and 1B illustrate first and second top views, respectively, ofa droplet actuator 100 in use during a first and second phase,respectively, of a droplet splitting operation. Droplet actuator 100makes use of an arrangement that is not suitably arranged forefficiently splitting a droplet using the specific technique shownwithout loss of magnetically responsive beads. Droplet actuator 100includes a first substrate 110 and a second substrate (not shown)arranged with a gap therebetween, which serves as a fluid path. Firstsubstrate 110 includes a set of droplet operation electrodes 112configured for conducting droplet operations on slug-shaped droplet 114,which is suspended in a filler fluid and includes magneticallyresponsive beads 116. A magnet 118 may be arranged in sufficientproximity to droplet operation electrodes 112 to permit some degree ofimmobilization of the magnetically responsive beads 116.

FIGS. 1A and 1B show a splitting operation that is taking place in thepresence of a magnetic field produced within droplet actuator 100 bymagnet 118. The placement of magnet 118 is not suitable for localizingsubstantially all of magnetically responsive beads 116 in a centralizedlocation (e.g., away from the edges of the droplet) within the portionof droplet 114 that is selected to retain the beads after the splittingoperation. Consequently, a certain quantity of magnetically responsivebeads 116 bridge splitting zone 120 during the droplet splittingoperation, as shown in FIG. 1A, and a loss of beads results, as shown inFIG. 1B. FIG. 1B shows a first droplet 122 that contains a certainquantity of the original quantity of magnetically responsive beads 116and a second droplet 124 that contains a certain remaining quantity ofthe original quantity of magnetically responsive beads 116. In otherwords, the end result of the illustrated splitting operation is a lossof magnetically responsive beads 116. The inventors have discovered thata contributing factor to the loss of beads is that a full quantity ofmagnetically responsive beads 116 is not suitably attracted,immobilized, and retained at a centralized location within droplet 114and/or at a sufficient distance from splitting zone 120.

8.2 Magnet Configurations for Preventing/Reducing Bead Loss

Among other things, the invention provides improved droplet actuatorsthat have various magnet configurations in which magnets are arrangedfor efficiently splitting bead-containing droplets and washingmagnetically responsive beads are described with reference to FIGS. 2,3, 4, 5A, and 5B. These figures illustrate nonlimiting examples ofmagnet configurations in combination with a droplet actuator splittingdroplets with little or no bead loss, and are, among other things,useful for efficiently washing magnetically responsive beads. One ormore magnets may be arranged in proximity to a droplet on a dropletactuator such that magnetically responsive beads are suitably attractedand immobilized within the droplet, preferably at a centralized locationaway from the neck that forms during splitting operations. In thisapproach, all or substantially all magnetically responsive beads areretained within a single droplet upon completion of a droplet splittingoperation. Similarly, the spitting operation may be performed in adroplet slug which is at a distance from the immobilized beads which issufficient to reduce or eliminate bead loss. As will be explained withrespect to the examples below, one or more magnets may be arranged withrespect to the droplet actuator structure above, below, and/or beside,the magnetically-responsive bead-containing droplet and any combinationsthereof to achieve this purpose.

8.2.1 Position of Magnet Relative to Splitting Zone

FIGS. 2A and 2B illustrate side and top views of a droplet actuator 200.In this embodiment, the magnet is placed at a position which issufficiently distant from the portion of the droplet which is breakingduring the splitting operation, splitting zone 224 (or vice versa, thesplitting zone may be said to be placed at sufficient distance from themagnet position), to reduce or eliminate bead loss during a dropletsplitting operation. Further, the magnet is positioned so that the beadsare generally centrally located along a lateral diameter L of thedroplet (top view). Droplet actuator 200 includes a first substrate 210and a second substrate 212 separated to province a gap for conductingdroplet operations, though only one substrate is required. A set ofdroplet operation electrodes 214 is associated with one or both of thesubstrates and arranged for conducting one or more droplet operations.Droplet actuator 200 may include a magnet 216 that is arranged insufficient proximity to droplet 218/222 to substantially immobilizemagnetically responsive beads 220 during a droplet splitting operation.For example, the magnet may be arranged as a component of the dropletactuator and/or in sufficient proximity to the droplet actuator toimmobilize the magnetically responsive beads in droplet 218/222 in thegap. Droplet 218/222 may be surrounded by a filler fluid (not shown).Droplet 218/222 contains a quantity of magnetically responsive beads 220immobilized by magnet 216.

Magnet 216 is positioned relative to one or more droplet operationelectrodes 214, in order to localize beads 220 in a region of theportion of the droplet 218/222 that is to form droplet 218 withoutpermitting substantial loss of beads 218 during the droplet splittingoperation to droplet 222.

In operation, a splitting operation is achieved without substantial lossof magnetic beads 220 by: as shown in 201, providing a droplet actuator200 with electrodes activated (ON) to form combined droplet 218/222 andmagnet 216 is arranged in a position which causes substantially all ofmagnetically responsive beads 220 to be attracted to magnet 216 in azone of droplet 218/222 that prevents substantial loss of magneticallyresponsive beads 220 to droplet 222. Magnet 216 may be arranged so thatmagnetically responsive beads 220 attracted thereto are localized at agenerally centralized location along lateral diameter L within combineddroplet 218/222 and away from the droplet split zone 224. During adroplet splitting operation as shown in 202, an intermediate electrodeis deactivated (OFF) to cause splitting at split zone 224. Substantiallyall magnetically responsive beads 220 are retained in droplet 218, anddroplet 222 is formed and is substantially free of magneticallyresponsive beads 220, as shown in 203.

A process for washing magnetically responsive beads 20 may, in oneembodiment, involve the repetition of droplet merging (with a washdroplet), bead immobilization, splitting, and bead resuspensionoperations until acceptable levels of washing are achieved.

8.2.2 Two-Magnet Arrangement to Produce Bead Column

FIG. 3 illustrates a side view of a droplet actuator 300. Dropletactuator 300 is generally configured as described is FIG. 2, except thatit includes two magnets, magnet 310 a and 310 b, arranged below andabove droplet 218. The magnets may be integral with droplet actuator 300and/or arranged in close proximity to the outer side of first substrate210 and second substrate 212. In general, the magnets 310 a and 310 bmay be arranged such that opposite poles are facing one another. In oneexample, the north or positive pole of magnet 310 a is facing the southor negative pole of magnet 310 b, as shown in FIG. 3.

Magnets 310 a and 310 b may be separate magnets or, alternatively,magnets 310 a and 310 b may be opposite poles of a single U-shaped,C-shaped, or horseshoe-shaped permanent magnet or electromagnet. Thearrangement of magnets 310 a and 310 b may cause magnetically responsivebeads 220 to be immobilized and retained in a column-shaped duster. Themagnets are preferably arranged to localize beads within droplet 218 ina position which is away form splitting zone 224 in the portion of thecombined droplet (not shown) in which the beads are to be retained.Further, the magnets are preferably aligned to centrally localize thebeads along lateral diameter L within the combined droplet (not shown).

8.2.3 Multiple Magnet Pairs to Centralize Beads

FIGS. 4A and 4B illustrate side views of a droplet actuator 400. Dropletactuator 400 is generally configured like droplet actuator 200 describedin FIG. 2, except that it includes four magnets arranged at positionssurrounding the droplet. In general, the arrangement illustrates theembodiment in which multiple magnet pairs with positive/negative polesfacing each other are arranged to generally centrally locate beads 220in the combined droplet prior to splitting. As illustrated in FIG. 4B,the beads are generally centrally located along a vertical dimension Vand a lateral dimension L. In the example illustrated, droplet actuator400 includes four magnets, such as a magnet 410 a, 410 b, 410 c, and 410d.

Magnets 410 a and 410 b may be arranged in close proximity to thedroplet, equally spaced on either side of the droplet, with oppositepoles facing each other. For example, the north pole of magnet 410 a mayface the south pole of magnet 410 b. Magnets 410 c and 410 d may bearranged in close proximity to the droplet, equally spaced on eitherside of the droplet, with opposite poles facing each other. For example,the north pole of magnet 410 d may face the south pole of magnet 410 c.Magnet pair 410 a/410 b may generally be aligned at right angles aroundthe droplet relative to magnet pair 410 c/410 d. In the illustratedembodiment, magnet pair 410 a/410 b has a vertical orientation aroundthe droplet, and magnet pair 410 c/410 d has a horizontal orientationaround the droplet. Any orientation around the droplet achieving thegenerally central localization of beads along lateral dimension L andvertical dimension V will suffice to achieve the desired centralimmobilization.

Magnets 410 a and 410 b may be arranged in close proximity to the outerside of first substrate 210 and second substrate 212, respectively, suchthat the magnetic field of magnets 410 a and 410 b may pass through thegap between first substrate 210 and second substrate 212 of dropletactuator 400. magnets 410 a and 410 b are arranged such that oppositepoles are facing one another. In one example, the north pole of magnet410 a is facing the south pole of magnet 410 b, as shown in FIG. 4.Similarly, magnets 410 c and 410 d are arranged in close proximity to afirst side and a second side, respectively, of droplet actuator 400,such that the magnetic field of magnets 410 c and 410 d may pass throughthe gap of droplet actuator 400 and perpendicular to the magnetic fieldof magnets 410 a and 410 b. Magnets 410 c and 410 d are arranged suchthat opposite poles are facing one another. In one example, the northpole of magnet 410 d is facing the south pole of magnet 410 c, as shownin FIG. 4.

Magnets 410 a and 410 b may be separate magnets or, alternatively,magnets 410 a and 410 b may be opposite poles of a single U-shaped,C-shaped, or horseshoe-shaped permanent magnet or electromagnet.Similarly, magnets 410 c and 410 d may be separate magnets or,alternatively, magnets 410 c and 410 d may be opposite poles of a singleU-shaped, C-shaped, or horseshoe-shaped permanent magnet orelectromagnet. Because the magnetic field of magnets 410 a and 410 b andmagnets 410 c and 410 d, respectively, intersect at the center of thefluid path within droplet actuator 400, the magnetically responsivebeads 220 are magnetically immobilized and retained in a cluster that iscentralized within the combined droplet and retained in droplet 218following the splitting operation.

8.2.4 Illustration of Splitting with Substantially No Bead Loss

FIGS. 5A and 5B illustrate a first and second top view, respectively, ofa droplet actuator 500 during a first and second phase, respectively, ofa droplet splitting operation. Droplet actuator 500 may alternatively beconfigured like any of the example droplet actuators 200, 300, and 400.Droplet actuator 500 of FIGS. 5A and 5B makes use of magnet forces thatare suitably arranged for use in a splitting operation designed toresult in substantially complete retention of beads in a single droplet,such as a process for washing magnetically responsive beads.

In particular, FIGS. 5A and 5B show a splitting operation that is takingplace at a sufficient distance from localized beads 220 to permit asplitting operation that results in substantially complete retention ofmagnetically responsive beads in droplet 218 and a droplet that issubstantially free of magnetically responsive beads. The position ofmagnet face 510 is suitably arranged to magnetically immobilizesubstantially all of magnetically responsive beds 220 at a centralizedlocation within the droplet and at a distance from the splitting zonethat is sufficient to achieve the desired retention of magneticallyresponsive beads 220 in droplet 218. As a result, substantially noquantity of magnetically responsive beads 220 bridges a splitting zone512 during the droplet splitting operation, as shown in FIG. 5A, andsubstantially no loss of beads occurs, FIG. 5B shows droplet 218 thatcontains substantially all magnetically responsive beads 220. In otherwords, the end result of a splitting operation that takes placesubstantially outside of the magnet forces is that there issubstantially no loss of magnetically responsive beads 220 becausesubstantially all of magnetically responsive beads 220 are suitablyattracted, immobilize, and retained at a centralized location within thefluid.

8.3 Droplet Actuator Configurations with Magnets 8.3.1 Droplet Actuatorwith Magnetic Shield

FIG. 6 illustrates a side view of a droplet actuator 600 that includes amagnetic shield for reducing the crossover of magnetic fields. Dropletactuator 600 includes a top plate 610 and a bottom plate 614 that arearranged having a gap 618 therebetween. An arrangement of electrodes622, e.g., electrowetting electrodes, may be associated with bottomplate 614 for performing droplet operations and an electrode, such asreservoir electrode 626 that is associated with a fluid reservoir 630that contains a quantity of fluid 634. One or more droplets (not shown)may be dispensed from the quantity of fluid 634 of reservoir 630 formanipulation along electrodes 622. Additionally, fluid 63 and anydroplets dispensed therefrom may optionally contain beads (not shown),which may, in some cases, be magnetically responsive.

Droplet actuator 600 further includes a magnet 638 that is arranged inproximity to the one or more electrodes 622. Magnet 638 may be arrangedin sufficient proximity to electrodes 622 in order to permitimmobilization of magnetically responsive beads (not shown), e.g., in adroplet positioned on an electrode. In one example, the purpose ofmagnet 638 is to magnetically immobilize and retain magneticallyresponsive beads during a droplet splitting operation, e.g., a splittingoperation that may be performed in a process for washing magneticallyresponsive beads.

Additionally, droplet actuator 600 includes a magnetic shield 642 thatis arranged in sufficient proximity to fluid reservoir 630 to shield thecontents thereof from nearby magnetic fields, such as the magnetic fieldof magnet 638. Magnetic shield 642 may, for example, be formed ofMu-metal that has sufficiently high magnetic permeability and that issuitable to reduce, preferably substantially eliminate, unwantedmagnetic fields from the magnet within fluid reservoir 630. In oneexample, magnetic shield 642 may be formed of Mu-metal that is suppliedby McMaster-Carr (Elmhurst, Ill.). Other examples of magnetic shield 642materials include Permalloy, iron, steel and nickel.

Droplet actuator 600 is not limited to one magnetic shield and onemagnet only, any number of magnetic shields and magnets may be installedtherein. Therefore, by use of one or more magnetic shields, the exposureof magnetic beads (not shown) within droplets to magnetic fields may belimited to desired regions only of droplet actuator 600. Magneticshields may be included on any surface of the droplet actuator and inany arrangement which facilitates suitable shielding.

In one example application, a droplet actuator may be employed toperform multiple assays in parallel and, consequently, there may be aneed for generally simultaneous washing of the various magnetic beadsthat may be manipulated within multiple lanes of electrodes. Withoutmagnetic shields, a wash operation or assay that is performed at acertain location using an associated magnet may be affected by themagnetic field of a distant magnet (i.e., crossover of magnetic fields).By contrast, crossover of magnetic fields between any two magnets may bereduced, preferably substantially eliminated, via the strategicplacement of one or mare magnetic shields, such as magnetic shield 642,within the droplet actuator.

8.3.2 Droplet Actuator with Alternating Magnet Configuration

FIG. 7 illustrates a top view of a droplet actuator 700 that includes amagnet whose poles are oppositely arranged for reducing the crossover ofmagnetic fields. Droplet actuator 700 includes an arrangement ofelectrodes 710, e.g., electrowetting electrodes, for performing dropletoperations on one or more droplets (not shown). Additionally, a magnet714 is arranged in close proximity to a first lane of electrodes 710, amagnet 718 is arranged in close proximity to a second lane of electrodes710, a magnet 722 is arranged in close proximity to a third lane ofelectrodes 710, and a magnet 726 is arranged in close proximity to afourth lane of electrodes 710. Magnets 714, 718, 722, and 726 may bearranged in sufficient proximity in electrodes 710 in order to permitimmobilization of magnetically responsive beads (trot shown) within oneor more droplets (not shown) located on one or more of the electrodes.

In order to reduce, preferably substantially eliminate, crossover ofmagnetic fields between adjacent magnets, the poles of adjacent magnetsare oppositely arranged, which causes the adjacent magnetic fields tocancel. For example and referring again to FIG. 7, the north pole ofmagnet 722 is oriented upward, and the south pole of magnet 726 isoriented upward. In this way, the magnetic fields are cancelled and thecrossover of magnetic fields between magnets 714, 718, 722, and 726 maybe reduced, preferably substantially eliminated.

FIG. 8 illustrates a map 800 of magnetic fields arranged in arelationship such as the one shown in FIG. 7.

8.4 Other Techniques

The invention also provides techniques for reducing carryover in adroplet actuator, as well as techniques for improving detectionoperations.

8.4.1 Technique for Reducing Carryover in a Droplet Actuator

FIG. 9 illustrates a top view of a droplet actuator 900 by which anoperation to reduce carryover at a droplet detection region may beperformed. Droplet actuator 900 includes an arrangement of electrodes910, e.g., electrowetting electrodes, for subjecting droplets to dropletoperations. Additionally, droplet actuator 900 includes a designateddetection region 914 at, for example, a certain electrode 910. Detectionregion 914 is used to detect droplets positioned thereon or passingtherethrough during droplet operations. In one example, dropletdetection is performed using a photomultiplier tube (PMT) orphoton-counting PMT that is associated with detection region 914. A PMT(not shown) is used to measure light (i.e., detect photons) emitted froma droplet (e.g., due to fluorescence and/or chemiluminescence) when atthe electrode that is associated with detection region 914.

In some cases, a build up of substances at a detection region, such asdetection region 914, may occur due to carryover, which involves beadsor other substances being left behind on surfaces and/or in filler fluidduring droplet operations. Carryover may interfere with accuratedetection of signals from subsequent droplets and/or interfere withdroplet operations by affected electrodes.

Referring again to FIG. 9, a droplet sequencing operation of theinvention reduces, preferably substantially eliminates, carryover atdetection region 914 by providing a series of alternating assay droplets918 and wash droplets 922. In one example, an assay droplet 918 a passesthrough detection region 914, followed by a wash droplet 922 a, followedby an assay droplet 918 b, followed by a wash droplet 922 b, followed byan assay droplet 918 c, followed by a wash droplet 922 c, followed by anassay droplet 918 d, which is followed by a wash droplet 922 d. As assaydroplets 918 a, 918 b, 918 c, and 918 d have the potential to degradethe function of detection region 914 due to crossover, wash droplets 922a, 922 b, 922 c, and 922 d perform a cleaning operation of the surfacesthat are associated with detection region 914. The cleaning process ofthe invention is not limited to the sequence that is shown in FIG. 9.Any sequence is possible as long as the sequence includes a suitablenumber of wash droplets to order to suitably clean the detection region.For example, depending on the requirements of the specific assay,multiple wash droplets may be provided between assay droplets and/ormultiple assay droplets may be provided between wash droplets, e.g.,AAWAAWAAW, AAAWAAAWAAAW, AWWAWWAWW, AWWWAWWWAWWW, AAWWAAWWAAWW,AAAWWWAAAWWWAAAWWW, etc., where A=as say droplet and W=wash droplet. Itshould be noted that it is not necessary for assay and wash droplets tobe the same size. Assay droplets may be larger or wash droplets may belarger. The larger droplet may be subjected to droplet operations as aslug (e.g., a slug occupying 4 electrodes) or as a single large droplet(e.g. a 4.times. droplet occupying as many electrodes as it naturallycovers without being formed into a slug). Each arrangement may result ina different cleaning result. It is also not necessary for the assaydroplets and wash droplets to follow the same path. For example, theirpaths may intersect at the location needing to be cleaned.

FIGS. 10A and 10B illustrate a top view of a droplet actuator 1000 bywhich another operation for reducing carryover may be performed. Dropletactuator 1000 includes an arrangement of electrodes 1010, e.g.,electrowetting electrodes, for performing droplet operations on one ormore droplets, such as assay droplets 1014 and 1018 (FIG. 10A) and washdroplet 1022 (FIG. 10B). Additionally, a magnet 1026 is arranged inclose proximity to a certain electrode 1010. Magnet 1026 may be arrangedin sufficient proximity to the certain electrode 1010 in order to permitimmobilization of magnetically responsive beads within one or moredroplets, such as magnetic beads 1030 within assay droplet 1014.

During, for example, a droplet split operation, a quantity of“satellite” droplets may be left behind at the point at which the splitoccurs. For example and referring to FIG. 10A, a droplet split operationby which assay droplet 1014 is formed by splitting from assay droplet1018 may result in a certain quantity of satellite droplets 1034 beingleft behind upon a certain electrode 1010. Satellite droplets, such assatellite droplets 1034, may be the source of carryover (crosscontamination) from one droplet to another, which is not desired. FIG.10B illustrates that a wash droplet, such as wash droplet 1022, may betransported along electrodes 1010 subsequent to the assay operations of,for example, assay droplets 1018 and 1014 in order to capture satellitedroplets 1034 and transport them away prior the next assay operationoccurs. In this way, electrodes 1010 are cleaned between assayoperations. The cleaning process of the invention is not limited to thesequence that is shown in FIGS. 10A and 10B. Any sequence is possible aslong the sequence includes a suitable number of wash droplets in orderto suitably clean the electrodes.

8.4.2 Improving Detection Operations in a Droplet Actuator

FIGS. 11A, 11B, and 11C illustrate side views of droplet actuator 1100by which respective operations for improving the sensitivity of dropletdetection may be performed. Droplet actuator 1100 includes a top plate1110 and a bottom plate 1114 that are arranged having a gap 1118therebetween. An arrangement of electrodes 1122, e.g., electrowettingelectrodes, may be associated with bottom plate 1114 for performingdroplet operations on a droplet 1126. A PMT window 1130 may beassociated with top plate 1110, by which a PMT (not shown) is used tomeasure light (i.e., detect photons 1134) emitted from droplet 1126.

FIG. 11A shows a method of improving the sensitivity of dropletdetection by spreading out a droplet, such as droplet 1126, in order toincrease the surface area that is exposed to PMT window 1130 and, thus,increase the number of photons 1134 that may be detected. A droplet maybe spread linearly across one or more electrodes 1122 depending on thevolume of the droplet. In the case of a small-volume droplet, a bufferdroplet may be added to make the droplet larger, as long as losses dueto dilution by the buffer droplet are offset by the increased dropletarea exposed to the PMT. For example, FIG. 11B shows droplet 1126 thatis spread continuously across multiple electrodes 1122, which increasesthe number of photons 1134 that may reach PMT window 130 and may bedetected by the PMT.

FIG. 11C shows a scenario wherein droplet 1126 is split up into multipledroplets 1126, such as droplets 1126 a, 1126 b, 1126 c, and 1126 d thatare on multiple electrodes 1122. Again, the surface area that is exposedto PMT window 1130 is increased, which increases the number of photons1134 that may reach PMT window 1130 and may be detected by the PMT.Alternatively and referring again to FIGS. 11A, 11B, 11C, the spreadingof a droplet, such as droplet 1126, is not limited to linear spreadingonly. The droplet may be spread two dimensions, such as across a grid orarray or electrodes 1122, in order to increase the surfaced area that isexposed to PMT window 1130. Alternatively, one or more large-areaelectrodes may be provided, across which one or more droplets may bespread.

FIG. 12 illustrates a top view of a droplet actuator 1200 for improvingthe sensitivity of droplet detection. Droplet actuator 1200 includes anarrangement of electrodes 1210, e.g., electrowetting electrodes, forperforming droplet operations on multiple droplets 1214.

Additionally, droplet actuator 1200 may include a droplet detectionregion 1218 that has an associated PMT (not shown) for measuring lightthat is emitted from a certain droplet 1214 when present. In order toreduce, preferably substantially eliminate, the carryover of light froma distant droplet 1214 to droplet detection region 1218, a minimumdistance d is maintained in all direction between the outer perimeter ofdroplet detection region 1218 and any distant droplets 1214 withindroplet actuator 1200, as shown in FIG. 12. The minimum distance d issufficiently large to reduce, preferably substantially eliminate, thecarryover of light from a distant droplet 1214 to droplet detectionregion 1218. As a result, in a droplet actuator that includes multipledroplets 1214, a spacing is maintained during detection between thetarget droplet 1214 that is being measured and a distant droplet 1214,such that the carryover of light from the distant droplet 1214 todroplet detection region 1218 is reduced, preferably substantiallyeliminated. As a specific case the distance d is an integer multiple mof a unit electrode size, and the droplet detection may be conducted ona set of electrodes which are electrically connected as an m phased bus.Alternatively, FIG. 13 (described below) describes a scenario whereinthe real-estate with a droplet actuator is limited and, therefore,sufficient spacings between droplets, as described in FIG. 12, may notbe achieved.

In an alternative embodiment, cross-over from nearby droplets iseliminated by using optical elements, such as one or more lenses, whichfocus only light from the droplet being interrogated onto the sensor andeliminates signal from other droplets.

FIG. 13 illustrates a side view of a droplet actuator 1300 for improvingthe sensitivity of droplet detection. Droplet actuator 1300 includes atop plate 1310 and a bottom plate 1314 that are arranged having a gap1318 therebetween. An arrangement of electrodes 1322, e.g.,electrowetting electrodes, may be associated with bottom plate 1314 forperforming droplet operations on droplets, such as a droplet 1326 and adroplet 1330. A PMT window 1334 may be associated with top plate 1310,by which a PMT (not shown) is used to measure light (i.e., detectphotons 1338) that is emitted from, for example, droplet 1326. Becausethe spacing between, for example, droplet 1326 at PMT window 1334 anddistant droplet 1330 is not suitably sufficient to avoid the carryoverof light from droplet 1330 to PMT window 1334, a mask 1342 is providedupon top plate 1310. The purpose of mask 1342 is to block light from adistant droplet from carrying over to PMT window 1334, which is thedetection region of a target droplet.

Mask 1342 may be formed upon top plate 1310 via a layer of anylight-absorbing material, as long as the material that is used iscompatible with the electrowetting process and does not unduly interferewith the droplet actuator operations. In one example, mask 1342 may beformed by applying a layer of black paint to top plate 1310, such thatone or more windows, such as PMT window 1334, are provided in selecteddefection regions of droplet actuator 1300. In the example shown in FIG.13, mask 1342 reduces, preferably substantially eliminates, the carryover of light from distant droplet 1330 to the target droplet 1320 atPMT window 1334. In another example the mask 1342 is formed by an opaqueconductor on the side of the top plated 1310 facing the droplet. Theconductor may, for example, be aluminum, chromium, copper, or platinum.The conductor may additionally serve as an electrical referenceelectrode.

8.5 Droplet Actuators with Magnet Assemblies

FIGS. 14A and 14B illustrate top views of a modular droplet actuatorassembly 1400, which is a nonlimiting example of a universal assemblyfor orienting a magnet assembly to a droplet actuator. Modular dropletactuator assembly 1400 include, for example, a mount 1410, a magnetassembly 1420, and a droplet actuator 1430, FIG. 14A shows modulardroplet actuate assembly 1400 when disassembled. FIG. 14B shows modulardroplet actuator assembly 1400 when assembled.

Magnet assembly 1420 may include a substrate 1424 upon which is mountedone or more magnets 1428, as shown in FIG. 14A. The magnets 1428 may bepermanently affixed to substrate 1424 or may be removable. Removablemagnets 1428 facilitate selection by a user of magnets having desiredproperties, such as desired magnet strength. In one embodiment, adroplet actuator instrument is provided with a droplet actuator assembly1400 including a mount 1410 and a magnet assembly 1420 without magnets.In another embodiment, the user is also provided with magnets havingspecified properties which may be affixed by the user to the magnetassembly 1420. In another embodiment, the user is also provided withsets of magnets having various specified characteristics such that theuser may select a set of one or more magnets having desired propertiesand affix the selected set to the magnet assembly 1420.

Magnets may be marked or coded (e.g., color coded) to facilitateselection of magnets having appropriate properties, as well as marked toshow the orientation of the magnet's magnetic field (e.g., by colorcoding or otherwise marking the North and South faces of the magnets).Similarly, the magnet assembly 1420 may be marked to show the desiredorientation of magnets inserted therein, and in some embodiments,magnets may be shaped to permit them to be affixed to the magnetassembly 1420 only in an appropriate orientation.

Moreover, in another embodiment, the user may be provided with magnetassemblies 1420 having magnets already affixed thereto, wherein themagnet assemblies 1420 each have different magnet configurations, e.g.,sets of magnets having different properties. The user may select themagnet configuration having magnets having properties appropriate to theuser's desired use for the instrument. Magnet assembly 1420 may bemarked or otherwise color coded to facilitate selection by the user.Magnet properties may, for example, be selected based on the propertiesof beads selected by the user.

Droplet actuator 1430 may include a substrate 1434 upon which is anarrangement of electrodes 1438, e.g., electrowetting electrodes, asshown in FIG. 14A. A second (top) substrate may also be included (notshown).

Magnet assembly 1420 is designed such that magnets 1428 substantiallyalign with certain electrodes 1438 of interest on droplet actuator 1430.For example, in some embodiments, parallel configurations of magnets maybe present for conducting parallel assay steps on droplet actuator 1430.Magnets may be configured and oriented, for example, according to thevarious configurations and orientations described herein.

Mount 1410 may serve as a universal platform for mounting a magnetassembly, such as magnet assembly 1420, and a droplet actuator, such asdroplet actuator 1430. In one embodiment, mount 1410 is configured toaccept a wide variety of magnet assemblies 1420 and a wide variety ofdroplet actuators 1430. Magnet assembly 1420 may include one or moremagnets arranged in any of a variety of patterns and employing any of avariety of magnet properties. FIG. 14 illustrates a row of magnets, butmagnets may also be provided in a grid or any arrangement that placesthe magnets in their proper position in relation to the droplet actuator1430 in order to facilitate desired operations on the droplet actuator.

In one example, magnet assembly 1420 and droplet actuator 1430 may beinstalled into mount 1410 via respective fittings 1418 and 1414.Fittings may, for example, involve slots into which the mount 1410 maybe fitted, openings on the mount 1410 for accepting posts on the magnetassemblies 1420 or vice versa, openings on the mount 1410 for acceptingscrews on the magnet assemblies 1420, threaded posts for acceptingbolts, various spring loaded mechanisms, recessed trays, complimentaryfittings, and the like. Any mechanism which facilitates sufficientlysecure attachment to permit the device to function for its intendedpurpose will suffice.

Further, the mount 1410 may include multiple fittings for multiplepossible positions of magnet assembly 1420 and/or droplet actuator 1430,and/or mounting of multiple magnet assemblies 1420 and/or multipledroplet actuators 1430 in a single mount 1410. Further, mount 1410 maybe configured to permit magnet assemblies 1420 to be mounted above,below and/or beside the droplet actuator 1430, i.e., in any relationshipwith the droplet actuator. With droplet actuator 1430 installed inmodular mount 1410, any magnet assembly of interest, such as magnetassembly 1420, may be inserted into modular droplet actuator assembly1400 via, for example, the slot.

FIG. 15 illustrates a side view of a modular droplet actuator assembly1500, which is another nonlimiting example of a universal assembly fororienting a magnet assembly to a droplet actuator, similar to theassembly 1400 shown in FIG. 14, except that mount 1410 is replaced witha mount 1510. Mount 1510 includes recessed trays into which magnetassembly 1420 and droplet actuator 1430 may be fitted in order toprovide a “drop in” method of loading.

Referring to FIGS. 14A, 14B, and 15, an aspect of the invention is thatthe slots or other attachment means serve to orient the magnet substrateand the droplet actuator so as to align the magnets on the magnetassembly with the appropriate electrodes or electrode paths on thedroplet actuator. In this manner, the magnets may be removed when notneeded for an assay. Additionally, different magnet mounts withdifferent distributions of magnets may be provided for different typesof assays or different droplet actuator layouts.

8.6 Magnets

In addition to other aspects already described, it should be noted thatmagnets selected for use with the invention may be permanent orelectromagnets. There may be a relationship between the magneticallyresponsive content of the beads at the droplet and the magneticstrength/pull force. Therefore, the magnet strength/pull force of themagnet may be selected relative to the responsiveness of the magnetbeads such that it is:

sufficiently strong relative to the magnetic responsiveness of the bendsto substantially immobilize magnetically responsive beads;

not so strong relative to the magnetic responsiveness of the beads thatit significantly magnetizes the beads and, thus, causes irreversibleformation of clumps of beads;

not so strong relative to the magnetic responsiveness of the beads thatresuspension occurs poorly when the magnet field is removed; and/or

not so strong relative to the magnetic responsiveness of the beads thatthe beads are pulled out of the droplet altogether.

In some embodiments, the magnet may have high magnetic strength (inTesla) with lesser pull force (in pounds). In one example, magnet is aneodymium permanent magnet that has a surface field strength of about 1Tesla (T). In another example, the magnet is an electromagnet that has asurface field strength of about 1 T, which may be turned on and offelectronically. Where a permanent magnet is used, the magnet may bemoved away from the magnetically responsive bead-containing droplet foruses in which it is desirable to remove the influence of the magneticfield. While not limited to the following ranges, it is understood thatranges of magnetic strength that generally encompasses the usefulstrength of the present invention can include: a broad range of 0.01 Tto 100 T (pulsed) or 45 T (continuous); an intermediate range of 0.01 Tto 10 T: and a narrow range of 0.1 T to 1 T (preferably 0.5 T).

8.7 Droplet Composition

Droplets including magnetic beads and subjected to droplet splittingoperations may include any of a wide variety of samples, reagents, andbuffers useful for conducting assays using the beads. For example,during washing, the droplet may include a buffer. such as aphosphate-buffered saline (PBS) buffer with a surfactant that issuitable for use in magnetic based immunoassays. Preferred surfactantsare these which facilitate immobilization and/or resuspension of beadsfollowing immobilization by magnetic forces. The surfactant and amountof surfactant may be adjusted to provide a substantial improvement insuspension as compared to a control solution lacking the surfactant. Inone embodiment, the droplet includes PBS buffer with about 0.01% Tween®20.

A hydrophilic polymer and/or surfactant may be included in the dropletto facilitate retention and resuspension of magnetically responsivebeads during splitting operation. The droplet may include a wide varietyof liquids immiscible with the filler fluid. Examples of buffersinclude, but are not limited to, phosphate-buffered saline (PBS) bufferand Tris buffer saline. In one embodiment, the droplet fluid includes abuffer, such as the PBS buffet; and any surfactant that is suitable foruse in magnetic based immunoassays.

Preferred hydrophilic polymers and surfactants are those whichfacilitate resuspension of beads following immobilization by magneticforces. The surfactant and amount of surfactant may be adjusted toprovide a substantial improvement in resuspension as compared to acontrol solution lacking the surfactant. Examples of surfactants thatare suitable for use in magnetic based immunoassays include, but are notlimited to, polysorbate 20, which is commercially known as Tween®20, andTriton X-100, Tween® 20 may be supplied by, for example, PierceBiotechnology, Inc. (Woburn, Mass.). Triton® X-100 may be supplied by,for example, Rohm & Haas Co (Philadelphia, Pa.). In one example, thedroplet fluid within the droplet actuator is a mix of PBS with Tween® 20in the range of from about 0.001% to about 0.1%. In another example, thedroplet fluid within the droplet actuator is a mix of PBS with about0.01% Tween® 20.

Other examples include pluronic surfactants, polyethylene glycol (PEG),methoxypolethylene glycol (MPEG), poly-sorbate (polyxoxyethylenesorbitan monocicates or Tween®), polyoxyethylene octy phenyl ether(Triton X-100®), polyvinyl pyrollidone, polyacrylic acid (andcrosslinked polyacrylic acid such as carbomer), polyglycosides (nonionicglycosidic surfactants such as octyl glucopyranoside) and solublepolysaccharides (and derivatives thereof) such as heparin, dextrans,methyl cellulose, propyl methyl cellulose (and other cellulose estersand ethers), dextrins, maltodextrins, glactomannans, arbainoglactans,beta glucans, alginates, agar, carrageenan, and plant gums such asxanthan gum, psyllium, guar gum, gum traganth, gun karya, gum ghatti andgum acacia. The particular additive can be selected for maximumcompatibility with a specific microfluidic sample.

8.8 Droplet Actuator

For examples of droplet actuator architectures that are suitable for usewith the present invention, see U.S. Pat. No. 6,911,132, entitled,“Apparatus for Manipulating Droplets by Electrowetting-BasedTechniques,” issued on Jun. 28, 2005, to Pamula et al.; U.S. patentapplication Ser. No. 11/343,284, entitled, “Apparatuses and methods forManipulating Droplets on a Printed Circuit Board,” filed on filed onJan. 30, 2006; U.S. Pat. No. 6,773,566, entitled, “ElectrostaticActuators for Microfluidics and Methods for Using Same,” issued on Aug.10, 2004 and U.S. Pat. No. 6,565,727, entitled, “Actuators forMicrofluidics Without Moving Parts,” issued on Jan. 24, 2000, both toShenderov et al., and International Patent Application No. PCT/US06/47486 to Pollack et al., entitled, “Droplet-Based Biochemistry,”filed on Dec. 11, 2006, the disclosures of which are incorporated hereinby reference. Droplet actuator techniques for immobilizing magneticbeads and/or non-magnetic beads are described in the foregoinginternational patent applications and in Sista, et al., U.S. PatentApplication No. 60/900,653, filed on Feb. 9, 2007, entitled“Immobilization of magnetically-responsive beads during dropletoperations”: Sista et al., U.S. Patent Application No. 60/969,736, filedon Sep. 4, 2007, entitled “Droplet Actuator Assay Improvements”; andAllen et al., U.S. Patent Application No. 60/957,717, filed on Aug. 24,2007, entitled “Bead washing using physical barriers,” the entiredisclosures of which is incorporated herein by reference. Combinationsof these various techniques are within the scope of this invention.

8.9 Fluids

For examples of fluids that may be subjected to droplet operations ofthe invention, see the patents listed in section 8.8, especiallyInternational Patent Application No. PCT/US 06/47486, entitled,“Droplet-Based Biochemistry,” filed on Dec. 11, 2006. In someembodiments, the droplet is a sample fluid, such as a biological sample,such as whole blood, lymphatic fluid, serum, plasma, sweat, tear,saliva, sputum, cerebrospinal fluid, amniotic fluid, seminal fluid,vaginal excretion, serous fluid, synovial fluid, pericardial fluid,peritoneal fluid, pleural fluid, transodates, exudates, cystic fluid,bile, urine, gastric fluid, intestinal fluid, fecal samples, fluidizedtissues, fluidized organisms, biological swabs and biological washes. Insome embodiments, the fluid that is loaded includes a reagent, such aswater, deionized water, saline solutions, acidic solutions, basicsolutions, detergent solutions and/or buffers. In some embodiments, thefluid that is loaded includes a reagent, such as a reagent for abiochemical protocol, such as a nucleic acid amplification protocol, anaffinity-based assay protocol, a sequencing protocol, and/or a protocolfor analyses of biological fluids.

8.10 Filler Fluids

As noted, the gap is typically filled with a filler fluid. The fillerfluid may, for example, be a low-viscosity oil, such as silicone oil.Other examples, of filler fluids are provided in International PatentApplication No. PCT/US 06/47486, entitled, “Droplet-Based Biochemistry,”filed on Dec. 11, 2006.

8.11 Washing Magnetically Responsive Beads

For protocols making use of beads, droplets with beads can be combinedusing droplet operations with one or more wash droplets. Then, whileretaining the beads (e.g., physically or magnetically) using magnetconfigurations of the invention, the merged droplet may be divided usingdroplet operations into two or more droplets: one or more droplets withbeads and one or more droplets without a substantial amount of beads. Inone embodiment, the merged droplet is divided using droplet operationsinto one droplet with beads and one droplet without a substantial amountof beads.

Generally, each execution of a washing protocol results in retention ofsufficient beads for conducting the intended assay without undulydetrimental effects on the results of the assay. In certain embodiments,each division of the merged droplet results in retention of more than90, 95, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99,99.9, 99.99, 99.999, 99.9999, 99.99999, or 99.999999 percent of beads.In other embodiments, each execution of a washing protocol to achieve apredetermined reduction in the concentration and/or amount of removedsubstance results in retention of more than 99, 99.1, 99.2, 99.3, 99.4,99.5, 99.6, 99.7, 99.8, 99, 99.9, 99.99, 99.999, 99.9999, 99.99999, or99.999999 percent of beads. In still other embodiments, the amount ofretained beads is calculated and the results are adjusted accordingly.

In some embodiments, beads can be washed in reservoirs in which thebead-containing droplet and wash droplets are combined, beads areretained (for example by a magnet, by physical structures, electrostaticforces), and droplets lacking beads are dispensed from the reservoirusing droplet operations. For example, beads can be washed bydilute-and-dispense strategy whereby a wash buffer is added to thereservoir to dilute the contents, magnetically responsive beads arelocalized within the reservoir with a magnet and moist of the solutionis dispensed front the reservoir, and this cycle is repeated tillacceptable levels of washing are achieved.

As an example, washing magnetically responsive beads may generallyinclude the following steps:

(1) providing a droplet comprising magnetically responsive beads andunbound substances in proximity with a magnet;

(2) using droplet operations to combine a wash droplet with the dropletcomprising the magnetically responsive beads;

(3) immobilizing the beads by application of a magnetic field;

(4) using droplet operations to remove some or all of the dropletsurrounding the beads to yield a droplet comprising the beads with areduced concentration of unbound components and a droplet comprisingunbound components;

(5) releasing the beads by removing the magnetic field; and

(6) repeating steps (2) to (3) or (2) to (4) until a predetermineddegree of purification is achieved.

In this manner, unbound substances, such as contaminants, byproducts orexcess reagents, can be separated from the beads. Each cycle produces adroplet including the beads but with a decreased level of the unwantedsubstances. Step (5) is not required in each washing cycle; however, itmay be useful to enhance washing by freeing contaminants which may betrapped in the immobilized beads. Steps may be performed in a differentorder, e.g., steps (2) and (3) may be reversed. Steps in the washingprotocol may be accomplished on a droplet actuator using dropletoperations as described herein.

In embodiments in which magnetically responsive beads are used, theinventors have found that application of a magnetic field though usefulfor temporarily immobilizing beads, moving beads and/or positioningbeads, sometimes results in unwanted aggregation of the beads. Asalready noted, in one embodiment, a hydrophilic polymer and/orsurfactant is included to prevent or reduce bead aggregation.Hydrophilic polymers and surfactants should be selected and used inamounts which reduce or eliminate bead aggregation and minimizenon-specific absorption while at the same time not resulting issignificant loss of target analytes or reagents from the droplet. In oneembodiment, the hydrophilic polymer and/or surfactant reduces beadclumping in a droplet in a non-gaseous filler fluid and specificallydoes not serve to reduce molecular adsorption of droplet components to asurface of the droplet actuator.

Another approach to eliminating or reducing clumping aggregation ofbeads involves the use of smaller numbers of larger beads. Any number ofbeads which can be contained in a droplet during one or more dropletoperations may be used. In some embodiments, the number of magneticallyresponsible beads can range from 1 to several 100,000's. For example, inone embodiment, the invention makes use of one to 100 magneticallyresponsible beads per droplet. For example, the invention may make useof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 . . . 100 magnetically responsive beadsper droplet. In one embodiment, the number of magnetically responsivebeads is from one to 10. Use of smaller numbers of magneticallyresponsive beads permits larger beads to be used. For example, in oneembodiment, the invention makes use of one to 100 magneticallyresponsive beads per droplet, where the beads have an average diameterof about 25 to about 100 microns. In another embodiment the inventionmakes use of one to 10 magnetically responsive beads per droplet, wherethe beads have an average diameter of about 50 to about 100 microns.

CONCLUDING REMARKS

The foregoing detailed description of embodiments refers to theaccompanying drawings, which illustrate specific embodiments of theinvention. Other embodiments having different structures and operationsdo not depart from the scope of the present invention.

This specification is divided into sections for the convenience of thereader only, Headings should not be construed as limiting of the scopeof the invention.

It will be understood that various details of the present invention maybe changed without departing from the scope of the present invention.Furthermore, the foregoing description is for the purpose ofillustration only, and not for the purpose of limitation, as the presentinvention is defined by the claims as set forth hereinafter.

1. A method comprising: effecting a change in a shape of a droplet,wherein the droplet is disposed over a substrate in sensing proximity toa sensor and the droplet has a starting surface area exposed to thesensor; and producing an expanded surface area of the droplet in thesensing proximity exposed to the sensor, wherein the expanded surfacearea exposed to the sensor is greater than the starting surface areaexposed to the sensor.
 2. The method of claim 1, further comprisingactivating one or more electrodes associated with the substrate.
 3. Themethod of claim 1, wherein the producing an expanded surface areacomprises an electrowetting mediated effect.
 4. The method of claim 1,wherein the sensor comprises a photon sensor, and the droplet comprisesa substance that emits photons.
 5. The method of claim 1, wherein thechange in the droplet's shape produces a slug.
 6. The method of claim 1,wherein the change in the droplet's shape produces a series ofsub-droplets.
 7. The method of claim 1, wherein in the expanded surfacearea of the droplet, the droplet is spread across multiple electrodes.8. The method of claim 1 wherein the droplet comprises one or moremagnetically responsive beads.
 9. A method comprising: activating one ormore electrodes associated with a substrate to effect a change in ashape of a droplet, wherein the droplet is disposed over a substrate insensing proximity to a photon sensor, and the droplet has a startingsurface area exposed to the sensor; and producing an expanded surfacearea of the droplet in sensing proximity to the photon sensor, whereinthe expanded surface area exposed to the sensor is greater than thestarting surface area exposed to the sensor.
 10. The method of claim 9wherein in the expanded surface area of the droplet, the droplet isspread across multiple electrodes.
 11. The method of claim 9, whereinthe droplet comprises one or more magnetically responsive beads.
 12. Themethod of claim 9, further comprising detecting photons emitted from thedroplet using a photomultiplier tube (PMT) or photon-counting PMTlocated in a droplet detection region of the substrate, wherein thephoton sensor comprises the photomultiplier tube (PMT) orphoton-counting PMT.
 13. A method comprising: transporting a firstdroplet from a set of two or more droplets into sensing proximity with asensor while retaining at least one remainder droplet of the set in abuffer zone; and transporting the first droplet away from the sensingproximity with the sensor and transporting a second droplet of the atleast one remainder droplet of the set from the buffer zone into thesensing proximity with the sensor.
 14. The method of claim 13 furthercomprising repeating the transporting with respect to one or moreadditional droplets, wherein the set has three or more droplets.
 15. Themethod of claim 13 wherein the first and second droplets are bufferedand transported on a droplet actuator.
 16. The method of claim 15,wherein: the droplet actuator comprises two substrates separated to forma gap; the droplets are positioned in the gap; the sensor is configuredin proximity to one of the substrates; and the sensing proximity of thesensor is limited by a mask on one or both of the substrates.
 17. Themethod of claim 16, further comprising detecting photons emitted fromthe droplet using a photomultiplier tube (PMT) or photon-counting PMTlocated in a droplet detection region of one of the substrates, whereinthe sensor comprises the photomultiplier tube (PMT) or photon-countingPMT.
 18. The method of claim 16, wherein the transporting of the firstdroplet and the transporting of the second droplet comprises the use ofa plurality of electrodes on one of the substrates.
 19. The method ofclaim 16, wherein the droplet comprises one or more magneticallyresponsive beads.
 20. The method of claim 19, wherein the dropletactuator further includes a magnet that is arranged in proximity to thearrangement of electrodes.